Particles comprising single stranded RNA and double stranded RNA for immunomodulation

ABSTRACT

The present invention relates to chimeric particles comprising single stranded RNA (ssRNA), double stranded RNA (dsRNA) and at least one cationic agent, a pharmaceutical composition containing said particles and to a method of producing the same. The particles of the present invention are particularly useful as an immunostimulating medicament with a superlative pattern of immunostimulation.

This application is a divisional of U.S. patent application Ser. No.14/361,827, issued May 2, 2017 as U.S. Pat. No. 9,636,414, and which wasfiled Jul. 8, 2014 under 35 U.S.C. 371 as a U.S. National Phaseapplication of International Patent Application No. PCT/EP2011/006358,and which was filed Dec. 15, 2011. The entirety of these applications isincorporated herein by reference.

The present invention relates to immunomodulating particles comprisingsingle stranded RNA and double stranded RNA, a pharmaceuticalcomposition comprising said particles and to a method of producing thesame. The particles of the present invention are particularly useful asimmunomodulating medicament with superlative and unexpected biologicalactivities.

BACKGROUND OF THE INVENTION

The immune system senses infections (e.g. virus or bacteria) as well aspathologic situations (e.g. necrosis) by the detection of danger signalstermed respectively PAMPs (Pathogens-Associated Molecular patterns) andDAMPs (Damage Associated Molecular Patterns). Those molecular signalsare detected by several families of specific receptors termed patternrecognition receptors (PRRs). PAMPS can be proteins (e.g. flagellin frombacteria), conjugated lipids such as lipopolysaccharides (LPS), ornucleic acids. In this later family, different forms of mislocalizednucleic acids are recognized by different PRRs present in endosomes:unmethylated CpG motifs in bacterial DNA are detected by Toll LikeReceptor (TLR)-9, single stranded RNA (ssRNA) is detected by TLR-7 andTLR-8 while double stranded RNA (dsRNA) is detected by TLR-3. Thosereceptors are expressed in distinct cell populations (Hornung V. et al.,2002, J. Immunol. 168, 4531-4537 and Jarrossay D. et al., 2001, Eur. J.Immunol. 31, 3388-3393) and trigger different intracellular signals thatresult in different types of immunostimulation i.e. induction ofdifferent types of cell surface activation markers and of differenttypes of cytokines. For example, stimulation through TLR-7 isparticularly efficacious to trigger interferon-alpha production whilestimulation through TLR-3 is particularly efficacious to triggerinterferon-beta production (Sandor F. and Buc, M., 2005, Folia Biologica(Praha) 51, 188-197). Of interest, pathogens and pathological situationscan trigger more than one PRR. For example Gram-negative bacteria canstimulate innate immunity through TLR-4 by LPS and also through TLR-9 byDNA. Thereby, several cell types get activated and consequentlycumulative immune responses can be triggered. This fact was overlookedand synthetic PAMP formulations developed with the goal of inducingtherapeutic immunomodulation to fight chronic diseases such aspersistent virus infections or cancer use one single PAMP. Using ssRNAit was recently reported that a clinically acceptable immunostimulatingformulation can be obtained by combining in specific conditions (i.e.low salts) the natural cationic peptides termed protamine and ssRNA(Rettig L. et al., 2010, Blood 115, 4533-4541 and WO 2009/144230). Thisformulation activates TLR-7 and TLR-8 but as expected not TLR-3.

The present invention is based on the observation that in adequateconditions ssRNA and dsRNA can be co-formulated into particles. Theimmunostimulation obtained by these ssRNA/dsRNA particles isunexpectedly higher than the immunostimulation obtained by particlescontaining each RNA individually. The production of bothinterferon-alpha and interferon-beta by human immune cells is higherusing the chimeric particles than when using respectively ssRNA- ordsRNA-containing particles. Thus, there is a synergy between thedifferent TLRs and/or different cell types resulting in some kind ofimmunobiological resonance that is of upmost interest for animmunomodulating drug.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a particlecomprising single stranded RNA (ssRNA) and double stranded RNA (dsRNA),wherein the RNA in the particle is associated with at least one cationicagent.

In one embodiment, the particle has a diameter in the range of fromabout 50 nm to about 1000 nm.

In one embodiment, the at least one cationic agent forms a complex withand/or encloses said RNA. In one embodiment, the at least one cationicagent is comprised in a vesicle enclosing the RNA, wherein the vesiclepreferably is a multilamellar vesicle, an unilamellar vesicle, or amixture thereof. In one embodiment, the vesicle is a liposome,preferably a cationic liposome. In one embodiment, the liposomecomprises a phospholipid such as phosphatidylcholine and/or a sterolsuch as cholesterol.

In one embodiment, the at least one cationic agent comprises apolycationic compound. In one embodiment, the at least one cationicagent comprises at least one agent selected from the group consisting ofan RNA-complexing lipid, an RNA complexing polymer and an RNA-complexingpeptide or protein. In one embodiment, the at least one cationic agentcomprises at least one agent selected from the group consistingprotamine, polyethyleneimine, a poly-L-lysine, a poly-L-arginine or ahistone.

In one embodiment, the at least one cationic agent comprises or consistof protamine. In this embodiment, the protamine:RNA (ssRNA+dsRNA) weightratio is from 16:1 to 1:2, more preferably from 4:1 to 1:2. A mass ratioof protamine to the total RNA (ssRNA+dsRNA) of 1 to 1 or higher (i.e.more protamine than total RNA) is preferred since it results in anoptimal immunostimulation.

In one embodiment, the ssRNA contains at least one U nucleotide and/orat least one G nucleotide. In one embodiment, the ssRNA is anoligonucleotide of from 6 to 100 nucleotides, preferably anoligonucleotide having the sequence according to SEQ ID NO: 1. In oneembodiment, the ssRNA is an mRNA of from 50 to 10,000 nucleotides.

In one embodiment, the dsRNA comprises two RNA molecules capable tofully or partially hybridize together. In one embodiment, the strands ofthe dsRNA are in average from 6 to 8000 nucleotides in length althoughthe benefits of the present invention are independent of the ssRNA anddsRNA lengths. In one embodiment, the dsRNA ispolyinosinic-polycytidylic acid (poly(I:C))

The particle of the invention may comprise a ligand for site specifictargeting such as an antibody. The ligand may be capable of binding to adisease-associated antigen such that the particle when administeredaccumulates at a diseased organ or tissue characterized by cellsexpressing the disease-associated antigen and preferably beingcharacterized by association of the disease-associated antigen withtheir cell surface, e.g. the disease-associated antigen is atransmembrane protein. The disease-associated antigen may be atumor-associated antigen and is preferably associated with the surfaceof a diseased cell such as a tumor cell but preferably not with thesurface of a healthy cell. Preferably the ligand for site specifictargeting binds to an extracellular portion of the disease-associatedantigen.

Particles of the invention when contacted with appropriate cells oradministered to a subject are capable of inducing interferon-alpha andinterferon-beta. Thus, the particles according to the invention areparticularly useful as an immunostimulating medicament.

The present invention also relates to a pharmaceutical compositioncomprising (the) particle(s) of the invention and optionally one or morepharmaceutically acceptable carriers, diluents and/or excipients. Thepharmaceutical composition of the invention may further comprise atleast one adjuvant such as an oil and/or at least one antigen.

The present invention also relates to a method for stimulating theimmune system of a subject comprising administering to the subject aneffective amount of a pharmaceutical composition of the presentinvention. The stimulation of the immune system preferably involves thestimulation of one or more of TLR-7, TLR-8 and TLR-3, preferably TLR-7and TLR-3, more preferably TLR-7, TLR-8 and TLR-3. Furthermore, thestimulation of the immune system preferably involves increasing thelevel of interferons, preferably interferon-alpha and/orinterferon-beta.

The pharmaceutical composition of the present invention may beco-administered with a further immunomodulating agent which may beselected from the group consisting of chemotherapeutic drugs,chloroquine, anti-CTLA-4 or anti-regulatory T-cell reagents and/or atleast one antigen. The immunomodulating agent and/or the at least oneantigen may be administered prior to, simultaneously with or afteradministration of the pharmaceutical composition of the presentinvention. If the immunomodulating agent and/or the at least one antigenis administered simultaneously with administration of the pharmaceuticalcomposition of the present invention, the immunomodulating agent and/orthe at least one antigen may be comprised in the pharmaceuticalcomposition of the present invention.

In a further aspect, the present invention provides an ex vivo methodfor stimulating immune cells by contacting the immune cells withparticles of the present invention. These stimulated immune cells can betransferred into a subject such as the subject from whom the immunecells were obtained to stimulate the immune system of the subject. Inone embodiment, suitable immune cells are isolated from a subject andare treated in vitro via adding to the isolated immune cells aneffective amount of particles of the present invention, and thestimulated immune cells are re-introduced into the subject. Suitableimmune cells for such ex vivo treatment include but are not limited todendritic cells and natural killer (NK) cells.

In a further aspect, the present invention relates to a method for theproduction of particles as defined herein, which method comprises thesteps of:

-   (a) providing an aqueous solution of ssRNA;-   (b) providing an aqueous solution of dsRNA;-   (c) providing an aqueous solution of protamine; and-   (d) combining the solutions obtained in steps (a) and (b) and mixing    it with the solution obtained in (c).

Preferably, the above steps (a) and (b) are performed by resuspending anappropriate amount of dried RNA in an aqueous solution containing 0 to125 mM electrolytes, preferably containing less than 100 mM, morepreferably less than 50 mM and, in particular, less than 25 mMelectrolytes.

Preferably, the above step (c) is carried out by diluting a solution ofprotamine, preferably an aqueous isotonic stock solution of protamine,preferably containing 1000 (“protamine 1000”) to 5000 (“protamine 5000”)heparin-neutralizing units per ml with a solution containing 0 to 125 mMelectrolytes, preferably containing less than 100 mM, more preferablyless than 50 mM and, in particular, less than 25 mM electrolytes. Forexample, protamine 1000 and 5000 stock solutions are commerciallyavailable from Valeant Pharmaceuticals International, Aliso Viejo,Calif., USA, under the trademarks Valeant® 1000 and 5000, respectively.

In one embodiment, particles of the present invention comprisingprotamine, ssRNA and dsRNA are prepared by diluting all threeingredients to less than 5 mg/ml, preferably to 1 mg/ml or less in anaqueous solution containing 0 to 125 mM electrolytes, preferably in purewater. In one embodiment, (i) protamine is formulated as a 1 mg/mlsolution by diluting a pharmaceutical isotonic solution of at least 10mg/ml (protamine 5000) with pure water and (ii) a mixture of equal massamounts of dsRNA and ssRNA is formulated as a 1 mg/ml solution bydiluting dried RNA pellets in pure water and (iii) these preparation aremixed. It has been demonstrated according to the invention that suchprocedure forms homogenous particles.

In one embodiment, the method according to the present inventioncomprises the following steps:

-   (a) providing an aqueous solution of ssRNA at less than 5 mg/ml in    pure water;-   (b) providing an aqueous solution of dsRNA at less than 5 mg/ml in    pure water-   (c) providing an aqueous solution of protamine at less than 5 mg/ml    by diluting an aqueous isotonic stock solution containing 5000    heparin-neutralizing units of protamine per ml with pure water;-   (d) combining the solutions obtained in steps (a) and (b), and-   (e) combining the solutions obtained in steps (d) and (c).

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects, advantages and novel features of the present inventionwill become apparent from the following detailed description whenconsidered in conjunction with the accompanying drawings.

FIG. 1: Addition of protamine to premixed ssRNA and dsRNA allows togenerate chimeric protamine/ssRNA/dsRNA particles containing both thedsRNA and the ssRNA.

RNA and protamine were diluted to 1 mg/ml using pure water. 5 microgramsof RNA (ssRNA:RNA18 or dsRNA or a mixture of 2.5 micrograms of ssRNAwith 2.5 micrograms of dsRNA) were mixed with 10 micrograms ofprotamine, the solutions were centrifuged and the pellets wereresuspended in 10 microliters of water containing 0.5% sodium dodecylsulfate (SDS) and 2 microliters of 10 mg/ml proteinase K were added todigest protamine. The formulations were separated on a 2% native agarosegel.

The gel on the left shows an example of the results obtained with HMWdsRNA and the gel on the right shows an example of the results obtainedwith LMW dsRNA. “R18” is ssRNA alone, “HMW” is high molecular weightdsRNA, “LMW” is low molecular weight dsRNA, “R18+HMVV” is a mixture ofRNA18 and HMW dsRNA, “R18+LMW” is a mixture of RNA18 and LMW dsRNA. Itis demonstrated that both ssRNA and dsRNA alone or mixed together gettrapped in the particles (found in the pellets).

FIG. 2: Immunostimulation capacity of protamine/ssRNA/dsRNA particles asreflected by interferon-alpha production in PBMCs from a healthy humandonor.

Two hundred microliters (1 million of cells) of a human Peripheral BloodMononuclear Cells (PBMCs obtained by centrifugation of fresh blood on aficoll solution) preparation were added on top of one of the following:4 micrograms of protamine (“Prot”); 2 micrograms of ssRNA,oligonucleotide RNA18 (“R18”); 2 micrograms of High Molecular WeightdsRNA (“HMW”); 4 micrograms of protamine mixed with 2 micrograms ofssRNA (“Prot-R18”); 4 micrograms of protamine mixed with 2 micrograms ofHigh Molecular Weight dsRNA (“Prot-HMW”); 4 micrograms of protaminemixed with 1 microgram of ssRNA and 1 microgram of High Molecular WeightdsRNA (“Prot-R18-HMW”); 2 micrograms of Low Molecular Weight dsRNA(“LMW”); 4 micrograms of protamine mixed with 2 micrograms of LowMolecular Weight dsRNA (“Prot-LMW”); 4 micrograms of protamine mixedwith 1 microgram of ssRNA and 1 microgram of Low Molecular Weight dsRNA(“Prot-R18-LMW”). As negative control, PBMCs were cultured alone.

Chimeric particles containing ssRNA and dsRNA (high or low molecularweight) are immunostimulating. In general, interferon-alpha productionwas superior using stimulation with chimeric particles (ssRNA+dsRNA)than when using particles containing only ssRNA. The addition ofprotamine on dsRNA was not increasing (and rather decreasing the minimalinterferon-alpha induction observed in some experiments by naked dsRNA)its immunostimulating activity as judged by interferon-alpha production.

FIG. 3: Immunostimulation capacity of protamine/ssRNA/dsRNA particles asreflected by interferon-beta production in PBMCs from a healthy humandonor.

Two hundred microliters (1 million of cells) of a human PBMC preparationwere added on top of one of the following: 4 micrograms of protamine(“Prot”); 2 micrograms of ssRNA, oligonucleotide RNA18 (“R18”); 2micrograms of High Molecular Weight dsRNA (“HMW”); 4 micrograms ofprotamine mixed with 2 micrograms of ssRNA (“Prot-R18”); 4 micrograms ofprotamine mixed with 2 micrograms of High Molecular Weight dsRNA(“Prot-HMW”); 4 micrograms of protamine mixed with 1 microgram of ssRNAand 1 microgram of High Molecular Weight dsRNA (“Prot-R18-HMW”); 2micrograms of Low Molecular Weight dsRNA (“LMW”); 4 micrograms ofprotamine mixed with 2 micrograms of Low Molecular Weight dsRNA(“Prot-LMW”); 4 micrograms of protamine mixed with 1 microgram of ssRNAand 1 microgram of Low Molecular Weight dsRNA (“Prot-R18-LMW”). Asnegative control, PBMCs were cultured alone.

Chimeric particles containing ssRNA and dsRNA (high or low molecularweight) are immunostimulating. In general, interferon-beta productionwas superior using stimulation with chimeric particles (ssRNA+dsRNA)than when using particles containing only ssRNA. The addition ofprotamine on dsRNA was not increasing but rather decreasing itsimmunostimulating activity as judged by interferon-beta production.Thus, it is unexpected that dsRNA increases the immunostimulatingactivity when incorporated in ssRNA-Protamine particles.

FIG. 4: Immunostimulation capacity of protamine/ssRNA/dsRNA particles asreflected by interferon-alpha production in PBMCs from a healthy humandonor using different protamine/total RNA ratios.

Two hundred microliters (1 million of cells) of a PBMC preparation wereadded on top of one of the following: 1 microgram of ssRNA (RNA18) and 1microgram of High Molecular Weight dsRNA (“0 for 1”); 2 micrograms ofprotamine mixed with 1 microgram of ssRNA (RNA18) and 1 microgram ofHigh Molecular Weight dsRNA (“1 for 1”); 4 micrograms of protamine mixedwith 1 microgram of ssRNA (RNA18) and 1 microgram of High MolecularWeight dsRNA (“2 for 1”); 8 micrograms of protamine mixed with 1microgram of ssRNA (RNA18) and 1 microgram of High Molecular WeightdsRNA (“4 for 1”)

Addition of protamine on a mixture of ssRNA and dsRNA allows stimulationof immune cells at protamine-RNA mass ratios from 1-1 up to 4-1.

FIG. 5: Synergistic immunostimulating activity of ssRNA and dsRNA as faras interferon-alpha production by plasmacytoid DC is concerned dependson third cell type(s).

pDCs (BDCA-2 positive) were mixed with one of the following: 2micrograms of ssRNA (RNA18) with 8 micrograms of protamine (bothreagents at 1 mg/ml diluted in pure water). (“Prot-R18”); 2 microgramsof dsRNA (high molecular weight poly (I:C)) with 8 micrograms ofprotamine (both reagents at 1 mg/ml diluted in pure water).(“Prot-HMW”); 1 microgram of ssRNA (RNA18) and 1 microgram of dsRNA(poly(I:C)) with 8 micrograms of protamine (all reagents at 1 mg/mldiluted in pure water) (“Prot-R18-HMW”). The negative control is thesame pDC-enriched cell population cultured in the presence of 8micrograms of protamine (“Prot”).

The high interferon-alpha production in PBMCs induced by chimericparticles containing 1 microgram of ssRNA and 1 microgram of dsRNA isnot obtained in enriched pDCs cell suspension. Protamine-based particlescontaining 2 micrograms of ssRNA are superior in stimulating sorted pDCscompared to chimeric particles containing 1 microgram of ssRNA and 1microgram of dsRNA. Thus, the capacity of the protamine/ssRNA/dsRNAparticles to induce high interferon-alpha production in PBMC dependsprobably on the stimulation by those chimeric particles of other cellsin addition to pDCs.

FIG. 6: Immunostimulating capacity of protamine/ssRNA/dsRNA particlesversus protamine/ssRNA mixed with protamine/dsRNA as reflected byinterferon-alpha production.

Two hundred microliters (1 million of cells) of a PBMC preparation wereadded on top of one of the following: 2 or 4 micrograms of protamine(“2micProt” and “4micProt”, respectively); 2 micrograms of ssRNA,oligonucleotide RNA18 (“2micR18”); 2 micrograms of Low Molecular WeightdsRNA (“2micLMW”); 1 microgram of ssRNA, oligonucleotide RNA18 mixedwith 1 microgram of Low Molecular Weight dsRNA (“1micR18+1micLMW”); 4micrograms of protamine mixed with 2 micrograms of ssRNA(“4micProt+2micR18”); 4 micrograms of protamine mixed with 2 microgramsof Low Molecular Weight dsRNA (“4micProt+2micLMW”); 4 micrograms ofprotamine mixed with 1 microgram of R18 combined with 1 microgram of LowMolecular Weight dsRNA (“4micProt+1micR18+1micLMW”); A mixture of twoindependent formulations (“2micProt+1micR18+2micProt+1micLMW”): thefirst one containing 2 microliters of protamine mixed with 1 microgramof single stranded RNA and the second one containing 2 microliters ofprotamine mixed with 1 microgram of dsRNA. As negative control, PBMCswere cultured alone.

Chimeric particles containing both ssRNA and dsRNA are moreimmunostimulating than a mixture of two formulations containingprotamine+ssRNA and protamine+dsRNA.

DETAILED DESCRIPTION OF THE INVENTION

In the following, definitions will be provided which apply to allaspects of the present invention.

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kölbi, Eds.,(1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of biochemistry, cell biology,immunology, and recombinant DNA techniques which are explained in theliterature in the field (cf., e.g., Molecular Cloning: A LaboratoryManual, 2^(nd) Edition, J. Sambrook et al. eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step or group of members, integers orsteps but not the exclusion of any other member, integer or step orgroup of members, integers or steps although in some embodiments suchother member, integer or step or group of members, integers or steps maybe excluded, i.e. the subject-matter consists in the inclusion of astated member, integer or step or group of members, integers or steps.The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”), provided herein is intended merely to better illustrate theinvention and does not pose a limitation on the scope of the inventionotherwise claimed. No language in the specification should be construedas indicating any non-claimed element essential to the practice of theinvention.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

The present inventor surprisingly observed that particles containingsingle stranded RNA (ssRNA) and double stranded RNA (dsRNA) within thesame particle can be formed. They have superlative immunostimulationcapacities as compared to particles containing either ssRNA or dsRNA.The superlative features of the chimeric particles probably relies oncross talk between cells stimulated by the particles through TLR-3and/or TLR-7 and/or TLR-8. For generating highly immunostimulatingparticles the ssRNA molecules are preferably at least 10 residues inlength and contain U-residues and/or the dsRNA molecules are preferablybetween 10 and 8000 residues in average length and are preferably madeof poly (I:C). Furthermore, protamine is preferred as cationic carrieragent in the particles and the mass ratio of protamine to total RNA ispreferably at least 0.5 (preferably not more than twice more RNA thanprotamine). In preferred embodiments this ratio is 1 or higher (mostpreferred the same mass amount of protamine and total RNA or moreprotamine than total RNA is used), 2 or higher, 4 or higher andpreferably up to 16, more preferably up to 8.

A preferred procedure for the preparation of particles of the inventioncontaining protamine as the cationic agent comprises the steps ofdiluting protamine, ssRNA and dsRNA at concentrations of less thanapprox. 5 mg/ml, at best at 1 mg/ml or less using pure water or low saltsolution (preferably less than 125 mM electrolytes), mixing the two RNAsolutions and then adding protamine preferably in mass excess comparedto the whole RNA content.

In the context of the present invention, the term “RNA” relates to amolecule which comprises ribonucleotide residues and preferably beingentirely or substantially composed of ribonucleotide residues.“Ribonucleotide” relates to a nucleotide with a hydroxyl group at the2′-position of a β-D-ribofuranosyl group. The term “RNA” comprisesisolated RNA such as partially or completely purified RNA, essentiallypure RNA, synthetic RNA, and recombinantly generated RNA and includesmodified RNA which differs from naturally occurring RNA by addition,deletion, substitution and/or alteration of one or more nucleotides.Such alterations can include addition of non-nucleotide material, suchas to the end(s) of a RNA or internally, for example at one or morenucleotides of the RNA. Nucleotides in RNA molecules can also comprisenon-standard nucleotides, such as non-naturally occurring nucleotides orchemically synthesized nucleotides or deoxynucleotides. These alteredRNAs can be referred to as analogs or analogs of naturally-occurringRNA.

According to the invention, “ssRNA” means single-stranded RNA andincludes mRNA, tRNA, rRNA, snRNAs, and other ssRNAs. ssRNA may containself-complementary sequences that allow parts of the RNA to fold andpair with itself to form double helices. According to the inventionpreferred as ssRNA are synthetic oligonucleotides of 6 to 100,preferably 10 to 50, in particular 15 to 30 or 15 to 20 nucleotides ormessenger RNA (mRNA) of more than 50 nucleotides, preferably of 50 to10,000, preferably 100 to 5000, in particular 200 to 1000 nucleotides.

According to the present invention, the term “mRNA” means“messenger-RNA” and relates to a “transcript” which may be generated byusing a DNA template and may encode a peptide or protein. Typically, anmRNA comprises a 5′-UTR, a protein coding region, and a 3′-UTR. In thecontext of the present invention, mRNA may be generated by in vitrotranscription from a DNA template. The in vitro transcriptionmethodology is known to the skilled person. For example, there is avariety of in vitro transcription kits commercially available.

According to the invention, “dsRNA” means double-stranded RNA and is RNAwith two partially or completely complementary strands. The size of thestrands may vary from 6 nucleotides to 10000, preferably 10 to 8000, inparticular 200 to 5000, 200 to 2000 or 200 to 1000 nucleotides.According to the invention preferred dsRNA is polyinosinic-polycytidylicacid (poly(I:C)), a synthetic analog of dsRNA. Poly(I:C) is composed ofa strand of poly(I) annealed to a strand of poly(C).

The dsRNA is preferably a fully or partially (interrupted) pair of RNAhybridized together. It can be made for example by mixing polyinosinicand polycytidylic acid RNA molecules. It also can be made by mixingdefined fully or partially pairing non-homopolymeric RNA strands.

There is no specific ribonucleotide sequence requirement for the ssRNAand dsRNA molecules to be suitable for preparing an immunostimulatoryparticle according to the present invention. However, it is not excludedthat certain ssRNA or dsRNA sequences would provide best biologicalactivities. However, preferably, the ssRNA component should contain atleast 25% uridine residues.

Most preferably, the ssRNA is an oligonucleotide that has the followingsequence (written 5′ to 3′): 18merU: AGUGUUAUUCUUGUAUGG (SEQ ID NO: 1).

According to the invention, the stability of RNA may be modified asrequired. For example, RNA may be stabilized by one or moremodifications having stabilizing effects on RNA.

The term “modification” in the context of RNA as used according to thepresent invention includes any modification of RNA which is notnaturally present in said RNA.

In one embodiment of the invention, the RNA used according to theinvention does not have uncapped 5′-triphosphates. Removal of suchuncapped 5′-triphosphates can be achieved by treating RNA with aphosphatase.

The RNA according to the invention may have modified naturally occurringor synthetic ribonucleotides in order to increase its stability and/ordecrease cytotoxicity. For example, in one embodiment, in the RNA usedaccording to the invention 5-methylcytidine is substituted partially orcompletely, preferably completely, for cytidine. Alternatively oradditionally, in one embodiment, in the RNA used according to theinvention pseudouridine is substituted partially or completely,preferably completely, for uridine.

In one embodiment, the term “modification” relates to providing an RNAwith a 5′-cap or 5′-cap analog. The term “5′-cap” refers to a capstructure found on the 5′-end of an mRNA molecule and generally consistsof a guanosine nucleotide connected to the mRNA via an unusual 5′ to 5′triphosphate linkage. In one embodiment, this guanosine is methylated atthe 7-position. The term “conventional 5′-cap” refers to a naturallyoccurring RNA 5′-cap, preferably to the 7-methylguanosine cap (m⁷G). Inthe context of the present invention, the term “5′-cap” includes a5′-cap analog that resembles the RNA cap structure and is modified topossess the ability to stabilize RNA if attached thereto, preferably invivo and/or in a cell.

Providing an RNA with a 5′-cap or 5′-cap analog may be achieved by invitro transcription of a DNA template in the presence of said 5′-cap or5′-cap analog, wherein said 5′-cap is co-transcriptionally incorporatedinto the generated RNA strand, or the RNA may be generated, for example,by in vitro transcription, and the 5′-cap may be attached to the RNApost-transcriptionally using capping enzymes, for example, cappingenzymes of vaccinia virus.

The RNA may comprise further modifications. For example, a furthermodification of the RNA used in the present invention may be anextension or truncation of the naturally occurring poly(A) tail.

The term “stability” of RNA relates to the “half-life” of RNA.“Half-life” relates to the period of time which is needed to eliminatehalf of the activity, amount, or number of molecules. In the context ofthe present invention, the half-life of an RNA is indicative for thestability of said RNA.

Of course, if according to the present invention it is desired todecrease stability of RNA, it is possible to modify RNA so as tointerfere with the function of elements as described above increasingthe stability of RNA.

According to the present invention, RNA may be obtained by chemicalsynthesis or by in vitro transcription of an appropriate DNA template.The promoter for controlling transcription can be any promoter for anyRNA polymerase. Particular examples of RNA polymerases are the T7, T3,and SP6 RNA polymerases. A DNA template for in vitro transcription maybe obtained by cloning of a nucleic acid, in particular cDNA, andintroducing it into an appropriate vector for in vitro transcription.The cDNA may be obtained by reverse transcription of RNA. Preferablycloning vectors are used for producing transcripts which generally aredesignated transcription vectors.

The term “expression” is used according to the invention in its mostgeneral meaning and comprises the production of RNA and/or peptides orproteins, e.g. by transcription and/or translation. With respect to RNA,the term “expression” or “translation” relates in particular to theproduction of peptides or proteins. It also comprises partial expressionof nucleic acids. Moreover, expression can be transient or stable.

In the context of the present invention, the term “transcription”relates to a process, wherein the genetic code in a DNA sequence istranscribed into RNA. Subsequently, the RNA may be translated intoprotein. According to the present invention, the term “transcription”comprises “in vitro transcription”, wherein the term “in vitrotranscription” relates to a process wherein RNA, in particular mRNA, isin vitro synthesized in a cell-free system, preferably using appropriatecell extracts. Preferably, cloning vectors are applied for thegeneration of transcripts. These cloning vectors are generallydesignated as transcription vectors and are according to the presentinvention encompassed by the term “vector”.

The term “translation” according to the invention relates to the processin the ribosomes of a cell by which a strand of messenger RNA directsthe assembly of a sequence of amino acids to make a peptide or protein.

Particles of the present invention preferably have a defined averagesize (diameter) of about 50 to about 1000 nm, preferably about 100 nm toabout 700 nm, more preferably about 200 nm to about 500 nm.

The average “size” of the particles is generally the “design size” orintended size of the particles prepared according to an establishedprocess. Size may be a directly measured dimension, such as average ormaximum diameter, or may be determined by an indirect assay such as afiltration screening assay. Direct measurement of particle size istypically carried out by dynamic light scattering. As minor variationsin size arise during the manufacturing process, a variation up to 40% ofthe stated measurement is acceptable and considered to be within thestated size. Alternatively, microcarrier size may be determined byfiltration screening assays. For example, a particle preparation is lessthan a stated size, if at least 97% of the particles pass through a“screen-type” filter of the stated size.

Cationic agents contemplated for use as carriers in the presentinvention include any substances or vehicles with which RNA can beassociated, e.g. by forming complexes with the RNA or forming vesiclesin which the RNA is enclosed or encapsulated, preferably resulting inincreased stability of the RNA compared to naked RNA.

The carriers useful according to the invention include lipid-containingcarriers such as cationic lipids, liposomes and micelles, cationicpolymers such as DEAE dextran or polyethyleneimine and nanoparticles.

Cationic lipids may form complexes with negatively charged nucleicacids. Any cationic lipid may be used according to the invention.Cationic lipids and cationic polymers can be used to complex nucleicacids, thereby forming so-called lipoplexes and polyplexes,respectively, and these complexes have been shown to deliver nucleicacids into cells.

Liposomes are microscopic lipidic vesicles often having one or morebilayers of a vesicle-forming lipid, such as a phospholipid, and arecapable of encapsulating a drug. Different types of liposomes may beemployed in the context of the present invention, including, withoutbeing limited thereto, multilamellar vesicles (MLV), small unilamellarvesicles (SUV), large unilamellar vesicles (LUV), sterically stabilizedliposomes (SSL), multivesicular vesicles (MV), and large multivesicularvesicles (LMV) as well as other bilayered forms known in the art. Thesize and lamellarity of the liposome will depend on the manner ofpreparation and the selection of the type of vesicles to be used willdepend on the preferred mode of administration. Preferred injectableliposomes are those in the size range of 10-500, 20-400, 50-200, 50-150,50-120, 50-100, or 50-90 nm in diameter. Cationic liposomes arestructures that are made of positively charged lipids and areincreasingly being used in gene therapy due to their favourableinteractions with negatively charged nucleic acids and cell membranes.Cationic liposomes are also known as cationic lipoplexes. Liposomesshould not be confused with micelles and reverse micelles composed ofmonolayers. The lipid assembly may be combined with stabilizers.Non-limiting examples of stabilizers include cholesterol and similarmembrane active sterols, lipopolymers such as PEGylated lipids.

Formation of liposomes is not a spontaneous process. Lipid vesicles areformed when phospholipids such as lecithin are placed in water andconsequently form one bilayer or a series of bilayers, each separated bywater molecules, once enough energy is supplied. Liposomes may be formedusing standard methods such as the reverse evaporation method (REV), thedehydration-rehydration method (DRV), sonication or other suitablemethods. Liposomes can be created, for example, by sonicatingphospholipids in water. Low shear rates create multilamellar liposomes,which have many layers. Continued high-shear sonication tends to formsmaller unilamellar liposomes. In this technique, the liposome contentsare the same as the contents of the aqueous phase. Sonication isgenerally considered a “gross” method of preparation as it can damagethe structure of the drug to be encapsulated. Newer methods such asextrusion and Mozafari method are employed to produce materials forhuman use.

After liposome formation, the liposomes can be sized to obtain apopulation of liposomes having a substantially homogeneous size range,typically between about 10 and 500 nm.

Any suitable liposome-forming material can be used in the presentliposomes.

The liposomes can include a vesicle-forming lipid derivatized with ahydrophilic polymer to form a surface coating of hydrophilic polymerchains on the liposome surface.

In accordance with one embodiment of the invention the particles of theinvention comprise on their outer surface a targeting agent which canselectively or preferably deliver the particles to a target cellpopulation, and/or to a target organ or tissue. For example, liposomesbearing ligands can target receptors expressed on diseased cells. Thisligand-binding promotes efficient drug uptake into cells and enhancesefficacy. One targeting means which has been explored employs antibodiesattached covalently or through electrostatic interactions to particlesurfaces.

According to the invention, protamine is preferred as cationic carrieragent. The term “protamine” refers to any of various strongly basicproteins of relatively low molecular weight that are rich in arginineand are found associated especially with DNA in place of somatichistones in the sperm cells of various animals (as fish). In particular,the term “protamine” refers to proteins found in fish sperm that arestrongly basic, are soluble in water, are not coagulated by heat, andyield chiefly arginine upon hydrolysis. In purified form, they are usedin a long-acting formulation of insulin and to neutralize theanticoagulant effects of heparin.

According to the invention, the term “protamine” as used herein is meantto comprise any protamine amino acid sequence obtained or derived fromnative or biological sources including fragments thereof and multimericforms of said amino acid sequence or fragment thereof. Furthermore, theterm encompasses (synthesized) polypeptides which are artificial andspecifically designed for specific purposes and cannot be isolated fromnative or biological sources.

The protamine used according to the present invention can be sulfatedprotamine or hydrochloride protamine. In a preferred embodiment, theprotamine source used for the production of the particles of theinvention is protamine 5000 which contains protamine at more than 10mg/ml (5000 heparin-neutralizing units per ml) in an isotonic saltsolution and which is diluted as set forth above.

The particles of the invention preferably have a protamine:total RNAweight ratio from 16:1 to 1:2, preferably from 8:1 to 1:2, morepreferably from 4:1 to 1:2. In one embodiment, the lower range limit ofthe protamine:total RNA weight ratio is 1:1, preferably 2:1. ThessRNA:dsRNA ratio can largely vary and be between 100:1 to 1:100,preferably 10:1 to 1:10, more preferably 1:1.

The term “peptide” according to the invention comprises oligo- andpolypeptides and refers to substances comprising two or more, preferably3 or more, preferably 4 or more, preferably 6 or more, preferably 8 ormore, preferably 10 or more, preferably 13 or more, preferably 16 more,preferably 21 or more and up to preferably 8, 10, 20, 30, 40 or 50, inparticular 100 amino acids joined covalently by peptide bonds. The term“protein” preferentially refers to large peptides, preferably topeptides with more than 100 amino acid residues, but in general theterms “peptide” and “protein” are synonyms and are used interchangeablyherein.

In the context of the present invention the terms “salt(s)” and“electrolyte(s)” are used interchangeably and mean a compound that atleast partially dissociates into its respective counter ions in water.

According to the present invention, the term “mM electrolytes” means theconcentration in 10⁻³ mol per liter of the sum of all electrolytes(including inorganic salts such as NaCl, KCl, NaH₂PO₄, Na₂HPO₄, KH₂PO₄,K₂HPO₄, MgCl₂, MnCl₂, Na₂SO₄, K₂SO₄, MgSO₄ and salts such Tris-HCl,EDTA, Hepes, etc.) in the solutions used to resuspend or to dilute thessRNA and dsRNA stock solutions and in the solutions used to dilute aprotamine stock solution (such as protamine 1000 or 5000) before mixingthe components (i.e. (a), (b) and (c) as defined above).

It should be noted that, once the particles of the present invention areformed, the specific salt (or electrolyte) concentration conditions usedfor preparing the particles need not to be further maintained. Thus, theparticles can be further processed, e.g. eventually recovered bycentrifugation and diluted, dissolved or dispersed in a medium,preferably a pharmaceutically acceptable excipient, vehicle and/ordiluent, in particular in an isotonic medium such as saline, Ringer orRinger Lactate solution.

It is demonstrated herein that particles of the present invention have astrong immunostimulatory effect and are able to induce a nonspecificgeneral activation of the immune system. Accordingly, the presentinvention provides a method of immunostimulation, in particular forstimulating an immune response in a subject, preferably a mammal,especially a human, comprising the administration of an effective amountof a pharmaceutical composition according to the invention.

The particles and pharmaceutical composition of the present inventionare useful to activate or strengthen the immunity in certain diseasestates, in particular in the case of chronic diseases, such as cancer orinfectious diseases, in particular persistent virus infections. Thus,the particles and pharmaceutical composition of the present inventionare useful in the treatment of said disease states. The particles andpharmaceutical composition of the present invention are particularlysuitable for inducing production, or increasing the level ofinterferons, in particular interferon-alpha and/or interferon-beta.Thus, the particles and pharmaceutical composition of the presentinvention may be used to supplement interferon-alpha treatment and/orinterferon-beta treatment, or to increase interferon-alpha and/orinterferon-beta in a subject.

According to the invention, the term “disease” refers to anypathological state, including cancer diseases. Cancer (medical term:malignant neoplasm) is a class of diseases in which a group of cellsdisplay uncontrolled growth (division beyond the normal limits),invasion (intrusion on and destruction of adjacent tissues), andsometimes metastasis (spread to other locations in the body via lymph orblood). These three malignant properties of cancers differentiate themfrom benign tumors, which are self-limited, and do not invade ormetastasize. Most cancers form a tumor, i.e. a swelling or lesion formedby an abnormal growth of cells (called neoplastic cells or tumor cells),but some, like leukemia, do not. The term “cancer” according to theinvention comprises leukemias, seminomas, melanomas, teratomas,lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer,kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skincancer, cancer of the brain, cervical cancer, intestinal cancer, livercancer, colon cancer, stomach cancer, intestine cancer, head and neckcancer, gastrointestinal cancer, lymph node cancer, esophagus cancer,colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer,breast cancer, prostate cancer, cancer of the uterus, ovarian cancer andlung cancer and the metastases thereof. Examples thereof are lungcarcinomas, mamma carcinomas, prostate carcinomas, colon carcinomas,renal cell carcinomas, cervical carcinomas, or metastases of the cancertypes or tumors described above. The term cancer according to theinvention also comprises cancer metastases.

Examples of cancers treatable with the particles and pharmaceuticalcomposition of the present invention include malignant melanoma, alltypes of carcinoma (colon, renal cell, bladder, prostate, non-small celland small cell lung carcinoma, etc.), lymphomas, sarcomas, blastomas,gliomas, etc.

Malignant melanoma is a serious type of skin cancer. It is due touncontrolled growth of pigment cells, called melanocytes.

According to the invention, a “carcinoma” is a malignant tumor derivedfrom epithelial cells. This group represents the most common cancers,including the common forms of breast, prostate, lung and colon cancer.

Lymphoma and leukemia are malignancies derived from hematopoietic(blood-forming) cells.

A sarcoma is a cancer that arises from transformed cells in one of anumber of tissues that develop from embryonic mesoderm. Thus, sarcomasinclude tumors of bone, cartilage, fat, muscle, vascular, andhematopoietic tissues.

Blastic tumor or blastoma is a tumor (usually malignant) which resemblesan immature or embryonic tissue. Many of these tumors are most common inchildren.

A glioma is a type of tumor that starts in the brain or spine. It iscalled a glioma because it arises from glial cells. The most common siteof gliomas is the brain.

By “metastasis” is meant the spread of cancer cells from its originalsite to another part of the body. The formation of metastasis is a verycomplex process and depends on detachment of malignant cells from theprimary tumor, invasion of the extracellular matrix, penetration of theendothelial basement membranes to enter the body cavity and vessels, andthen, after being transported by the blood, infiltration of targetorgans. Finally, the growth of a new tumor, i.e. a secondary tumor ormetastatic tumor, at the target site depends on angiogenesis. Tumormetastasis often occurs even after the removal of the primary tumorbecause tumor cells or components may remain and develop metastaticpotential. In one embodiment, the term “metastasis” according to theinvention relates to “distant metastasis” which relates to a metastasiswhich is remote from the primary tumor and the regional lymph nodesystem.

Examples of infectious diseases treatable with the particles andpharmaceutical composition of the present invention include viralinfectious diseases, such as AIDS (HIV), hepatitis A, B or C, herpes,herpes zoster (chicken-pox), German measles (rubella virus), yellowfever, dengue etc. flaviviruses, influenza viruses, hemorrhagicinfectious diseases (Marburg or Ebola viruses), bacterial infectiousdiseases, such as Legionnaire's disease (Legionella), gastric ulcer(Helicobacter), cholera (Vibrio), infections by E. coli, Staphylococci,Salmonella or Streptococci (tetanus); infections by protozoan pathogenssuch as malaria, sleeping sickness, leishmaniasis; toxoplasmosis, i.e.infections by Plasmodium, Trypanosoma, Leishmania and Toxoplasma; orfungal infections, which are caused e.g. by Cryptococcus neoformans,Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidisor Candida albicans).

The particles and pharmaceutical composition of the present inventionare also useful in treating allergies.

The particles and pharmaceutical composition of the present inventioncan also be used in conjunction with another therapeutic agent which canbe administered prior to, simultaneously with or after administration ofthe particles or pharmaceutical composition of the present invention.Such therapeutic agents include chemotherapeutic drugs for cancerpatients, e.g. gemcitabine, etopophos, cis-platin, carbo-platin,antiviral agents, anti-parasite agents or an anti-bacterial agents and,if administered simultaneously with the particles of the presentinvention, may be present in a pharmaceutical composition of the presentinvention.

In particular, the particles and pharmaceutical composition of thepresent invention can also be used in conjunction with animmunotherapeutic agent, preferably an immunotherapeutic agent inducingor effecting a targeted, i.e. specific, immune reaction. Suchimmunotherapeutic agents include agents directed against adisease-associated antigen such as therapeutic antibodies or agentsinducing an immune response direct against a disease-associated antigenor cells expressing a disease-associated antigen. Usefulimmunotherapeutic agents include proteins or peptides inducing a B cellor T cell response against the disease-associated antigen or cellsexpressing the disease-associated antigen. These proteins or peptidesmay comprise a sequence essentially corresponding to or being identicalto the sequence of the disease-associated antigen or one or morefragments thereof. In one embodiment, the protein or peptide comprisesthe sequence of an MHC presented peptide derived from thedisease-associated antigen. Instead of administering the protein orpeptide it is also possible to administer nucleic acid, preferably mRNA,encoding the protein or peptide. Accordingly, the pharmaceuticalcomposition of the present invention may be used in genetic vaccination,wherein an immune response is stimulated by introduction into a subjecta suitable nucleic acid molecule (DNA or mRNA) which codes for anantigen or a fragment thereof.

If the nucleic acid to be administered as therapeutic agent is mRNA itcan be present in the particles of the invention in addition to thessRNA and dsRNA or it can form at least part of the ssRNA present in theparticles of the invention.

In one embodiment, a disease-associated antigen is a tumor-associatedantigen. In this embodiment, the particles and pharmaceuticalcomposition of the present invention may be useful in treating cancer orcancer metastasis. Preferably, the diseased organ or tissue ischaracterized by diseased cells such as cancer cells expressing adisease-associated antigen and/or being characterized by association ofa disease-associated antigen with their surface. Immunisation withintact or substantially intact tumor-associated antigen or fragmentsthereof such as MHC class I and class II peptides or nucleic acids, inparticular mRNA, encoding such antigen or fragment makes it possible toelicit a MHC class I and/or a class II type response and thus, stimulateT cells such as CD8+ cytotoxic T lymphocytes which are capable of lysingcancer cells and/or CD4+ T cells. Such immunization may also elicit ahumoral immune response (B cell response) resulting in the production ofantibodies against the tumor-associated antigen. Furthermore, antigenpresenting cells (APC) such as dendritic cells (DCs) can be loaded withMHC class I-presented peptides directly or by transfection with nucleicacids encoding tumor antigens or tumor antigen peptides in vitro andadministered to a patient.

According to the present invention, a tumor-associated antigenpreferably comprises any antigen which is characteristic for tumors orcancers as well as for tumor or cancer cells with respect to type and/orexpression level. In one embodiment, the term “tumor-associated antigen”relates to proteins that are under normal conditions, i.e. in a healthysubject, specifically expressed in a limited number of organs and/ortissues or in specific developmental stages, for example, thetumor-associated antigen may be under normal conditions specificallyexpressed in stomach tissue, preferably in the gastric mucosa, inreproductive organs, e.g., in testis, in trophoblastic tissue, e.g., inplacenta, or in germ line cells, and are expressed or aberrantlyexpressed in one or more tumor or cancer tissues. In this context, “alimited number” preferably means not more than 3, more preferably notmore than 2 or 1. The tumor-associated antigens in the context of thepresent invention include, for example, differentiation antigens,preferably cell type specific differentiation antigens, i.e., proteinsthat are under normal conditions specifically expressed in a certaincell type at a certain differentiation stage, cancer/testis antigens,i.e., proteins that are under normal conditions specifically expressedin testis and sometimes in placenta, and germ line specific antigens. Inthe context of the present invention, the tumor-associated antigen ispreferably associated with the cell surface of a cancer cell and ispreferably not or only rarely expressed in normal tissues. Preferably,the tumor-associated antigen or the aberrant expression of thetumor-associated antigen identifies cancer cells. In the context of thepresent invention, the tumor-associated antigen that is expressed by acancer cell in a subject, e.g., a patient suffering from a cancerdisease, is preferably a self-protein in said subject. In preferredembodiments, the tumor-associated antigen in the context of the presentinvention is expressed under normal conditions specifically in a tissueor organ that is non-essential, i.e., tissues or organs which whendamaged by the immune system do not lead to death of the subject, or inorgans or structures of the body which are not or only hardly accessibleby the immune system. Preferably, the amino acid sequence of thetumor-associated antigen is identical between the tumor-associatedantigen which is expressed in normal tissues and the tumor-associatedantigen which is expressed in cancer tissues. Preferably, atumor-associated antigen is presented in the context of MHC molecules bya cancer cell in which it is expressed.

Examples for differentiation antigens which ideally fulfill the criteriafor tumor-associated antigens as contemplated by the present inventionas target structures in tumor immunotherapy, in particular, in tumorvaccination are the cell surface proteins of the claudin family, such asCLDN6 and CLDN18.2. These differentiation antigens are expressed intumors of various origins, and are particularly suited as targetstructures in connection with antibody-mediated cancer immunotherapy dueto their selective expression (no expression in a toxicity relevantnormal tissue) and localization to the plasma membrane.

Further examples for antigens that may be useful in the presentinvention are p53, ART-4, BAGE, beta-catenin/m, Bcr-abL CAMEL, CAP-1,CASP-8, CDC27/m, CDK4/m, CEA, CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M,ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2/neu, HPV-E7, HPV-E6,HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1/Melan-A, MC1R,Myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190minor BCR-abL, Pm1/RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 orRU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN,TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2, TPTE and WT, preferably WT-1.

The pharmaceutical composition of the present invention may take theform of a vaccine preparation comprising the particles of the inventionand at least one antigen such as an antigen as discussed above or animmunogenic fragment thereof, or a nucleic acid, in particular RNA,encoding said antigen or fragment.

An “antigen” is to be understood as meaning any structure which cancause the formation of antibodies and/or the activation of a cellularimmune response. Examples of antigens are polypeptides, proteins, cells,cell extracts, carbohydrates/polysaccharides, polysaccharide conjugates,lipids, and glycolipids. These antigens may be tumor antigens or viral,bacterial, fungal and protozoological antigens or allergens. The term“antigen” also includes derivatized antigens as secondary substancewhich becomes antigenic—and sensitizing—only through transformation(e.g., intermediately in the molecule, by completion with body protein),and conjugated antigens which, through artificial incorporation ofatomic groups (e.g., isocyanates, diazonium salts), display a newconstitutive specificity. The antigen may be present in the vaccineaccording to the invention in the form of a hapten coupled to a suitablecarrier. Suitable carriers are known to those ordinarily skilled in theart and include e.g. human serum albumin (HSA), polyethylene glycols(PEG). The hapten may be coupled to the carrier by processes well-knownin the prior art, e.g. in the case of a polypeptide carrier via an amidebond to a Lys residue.

By “treat” is meant to administer a compound or composition as describedherein to a subject in order to prevent or eliminate a disease,including reducing the size of a tumor or the number of tumors in asubject; arrest or slow a disease in a subject; inhibit or slow thedevelopment of a new disease in a subject; decrease the frequency orseverity of symptoms and/or recurrences in a subject who currently hasor who previously has had a disease; and/or prolong, i.e. increase thelifespan of the subject.

In particular, the term “treatment of a disease” includes curing,shortening the duration, ameliorating, preventing, slowing down orinhibiting progression or worsening, or preventing or delaying the onsetof a disease or the symptoms thereof.

The term “immunotherapy” relates to a treatment preferably involving aspecific immune reaction and/or immune effector function(s).

The term “immunization” or “vaccination” describes the process oftreating a subject for therapeutic or prophylactic reasons.

The term “subject” relates to mammals. For example, mammals in thecontext of the present invention are humans, non-human primates,domesticated animals such as dogs, cats, sheep, cattle, goats, pigs,horses etc., laboratory animals such as mice, rats, rabbits, guineapigs, etc. as well as animals in captivity such as animals of zoos. Theterm “animal” as used herein also includes humans.

The pharmaceutical compositions of the invention are preferably sterileand contain an effective amount of the particles of the invention andoptionally of further agents as discussed herein such as therapeuticagents and antigens to generate the desired reaction or the desiredeffect.

The pharmaceutical composition of the invention may be formulated as anemulsion containing an oil such as Montanide®.

The pharmaceutical composition of the invention may also comprise anadditional immunomodulating agent such as anti-CTL-A4 or anti-regulatoryT-cell reagents such as an anti-CD25 antibody or cyclophosphamide.

The pharmaceutical composition of the invention may be administeredtogether with supplementing immunity-enhancing substances such as one ormore adjuvants and may comprise one or more immunity-enhancingsubstances to further increase its effectiveness, preferably to achievea synergistic effect of immunostimulation. The term “adjuvant” relatesto compounds which prolongs or enhances or accelerates an immuneresponse. Various mechanisms are possible in this respect, depending onthe various types of adjuvants. For example, compounds which allow thematuration of the DC, e.g. lipopolysaccharides or CD40 ligand, form afirst class of suitable adjuvants. Generally, any agent which influencesthe immune system of the type of a “danger signal” (LPS, GP96, dsRNAetc.) or cytokines, such as GM-CFS, can be used as an adjuvant whichenables an immune response to be intensified and/or influenced in acontrolled manner. CpG oligodeoxynucleotides can optionally also be usedin this context, although their side effects which occur under certaincircumstances, as explained above, are to be considered. Because of thepresence of the immunostimulating agent according to the inventioncomprising ssRNA and dsRNA as the primary immunostimulants, however,only a relatively small amount of CpG DNA is necessary (compared withimmunostimulation with only CpG DNA). Thus, CpG DNA could be added inthe ssRNA/dsRNA mixture before addition of protamine so that all nucleicacids are condensed within particles or added on preformedprotamine/ssRNA/dsRNA particles. Particularly preferred adjuvants arecytokines, such as monokines, lymphokines, interleukins or chemokines,e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12,INFα, INF-γ, GM-CSF, LT-α, or growth factors, e.g. hGH. Further knownadjuvants are aluminium hydroxide, Freund's adjuvant or oil such asMontanide®, most preferred Montanide® ISA51.

Lipopeptides, such as Pam3Cys, are also suitable for use as adjuvants inthe pharmaceutical composition of the present invention.

Pharmaceutical compositions are usually provided in a uniform dosageform and may be prepared in a manner known per se. The pharmaceuticalcomposition of the invention may e.g. be in the form of a solution orsuspension.

The pharmaceutical composition of the invention may comprise salts,buffer substances, preservatives, carriers, diluents and/or excipientsall of which are preferably pharmaceutically acceptable. The term“pharmaceutically acceptable” refers to the non-toxicity of a materialwhich does not interact with the action of the active component of thepharmaceutical composition.

Salts which are not pharmaceutically acceptable may used for preparingpharmaceutically acceptable salts and are included in the invention.Pharmaceutically acceptable salts of this kind comprise in a nonlimiting way those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic acids, and the like. Pharmaceuticallyacceptable salts may also be prepared as alkali metal salts or alkalineearth metal salts, such as sodium salts, potassium salts or calciumsalts.

Suitable buffer substances for use in the pharmaceutical composition ofthe invention include acetic acid in a salt, citric acid in a salt,boric acid in a salt and phosphoric acid in a salt.

Suitable preservatives for use in the pharmaceutical composition of theinvention include benzalkonium chloride, chlorobutanol, paraben andthimerosal.

An injectable formulation may comprise a pharmaceutically acceptableexcipient such as Ringer Lactate.

The term “carrier” refers to an organic or inorganic component, of anatural or synthetic nature, in which the active component is combinedin order to facilitate, enhance or enable application. According to theinvention, the term “carrier” also includes one or more compatible solidor liquid fillers, diluents or encapsulating substances, which aresuitable for administration to a patient.

Possible carrier substances for parenteral administration are e.g.sterile water, Ringer, Ringer lactate, sterile sodium chloride solution,polyalkylene glycols, hydrogenated naphthalenes and, in particular,biocompatible lactide polymers, lactide/glycolide copolymers orpolyoxyethylene/polyoxy-propylene copolymers.

The term “excipient” when used herein is intended to indicate allsubstances which may be present in a pharmaceutical composition of thepresent invention and which are not active ingredients such as, e.g.,carriers, binders, lubricants, thickeners, surface active agents,preservatives, emulsifiers, buffers, flavoring agents, or colorants.

The agents and compositions described herein may be administered via anyconventional route, such as by parenteral administration including byinjection or infusion. Administration is preferably parenterally, e.g.intravenously, intraarterially, subcutaneously, intradermally orintramuscularly.

Compositions suitable for parenteral administration usually comprise asterile aqueous or nonaqueous preparation of the active compound, whichis preferably isotonic to the blood of the recipient. Examples ofcompatible carriers and solvents are Ringer solution and isotonic sodiumchloride solution. In addition, usually sterile, fixed oils are used assolution or suspension medium.

The agents and compositions described herein are administered ineffective amounts. An “effective amount” refers to the amount whichachieves a desired reaction or a desired effect alone or together withfurther doses. In the case of treatment of a particular disease or of aparticular condition, the desired reaction preferably relates toinhibition of the course of the disease. This comprises slowing down theprogress of the disease and, in particular, interrupting or reversingthe progress of the disease. The desired reaction in a treatment of adisease or of a condition may also be delay of the onset or a preventionof the onset of said disease or said condition.

An effective amount of an agent or composition described herein willdepend on the condition to be treated, the severeness of the disease,the individual parameters of the patient, including age, physiologicalcondition, size and weight, the duration of treatment, the type of anaccompanying therapy (if present), the specific route of administrationand similar factors. Accordingly, the doses administered of the agentsdescribed herein may depend on various of such parameters. In the casethat a reaction in a patient is insufficient with an initial dose,higher doses (or effectively higher doses achieved by a different, morelocalized route of administration) may be used.

The following examples are intended to illustrate preferred embodimentsof the invention and should not be interpreted to limit the scope of theinvention as defined in the claims.

EXAMPLES Example 1 Method for the Preparation of an ImmunostimulatoryProtamine/ssRNA/dsRNA Particle Composition

An oligoribonucleotide of approximately 18 residues is synthesized andpurified. The product is then lyophilized and resuspended at 1 mg/ml inpure water. Polyinosinic acid and polycytidylic acid are chemicallysynthesized, purified and hybridized before being lyophilized andresuspended at 1 mg/ml in pure water. Protamine Valeant® 5000 is diluted14 times in pure water to provide a solution of protamine atapproximately 1 mg/ml in low salt. The oligoribonucleotide and thehybridized polyinosinic-polycytidylic acid are mixed at equal amounts.Then, 4 volumes of 1 mg/ml protamine are added. Immediate and intensivemixing is performed for example by pipetting up and down or byvortexing. The formulation is left for a few minutes on the bench andcan then be further diluted with injection solution (for example Ringerlactate) or mixed with the antigen and eventually Montanide®. Should theprotamine/ssRNA/dsRNA particle solution be too diluted, the particlescan be recovered by centrifugation or freeze drying and be resuspendedin the adequate volume of desired solution before being used fortreatment formulation.

Example 2 Raw Physical Data Obtained from Size and CompositionMeasurement Experiments for Protamine/ssRNA/dsRNA Compositions

The 18 mer U rich ssRNA oligonucleotide (RNA18: 5′ AGUGUUAUUCUUGUAUGG3′, (SEQ ID NO: 1); stock solution at 5 mg/ml in water, “R18”), highmolecular weight (HMW, average length of the RNA molecules is from 1500to 8000 base pairs) and Low Molecular Weight (LMW, average length of theRNA molecules is from 200 to 1000 base pairs) dsRNA (Poly(I:C) composedof a strand of poly(I) annealed to a strand of poly(C)) and ProtamineVALEANT® 5000 (14 mg/ml stock solution in an isotonic injectionsolution, “P”) were diluted to 1 mg/ml using water, 25 mM NaCl, 75 mMNaCl or 125 mM NaCl. A total of 5 micrograms of RNA (either 5 microgramsof one species of RNA or a mixture of 2.5 micrograms of ssRNA and 2.5micrograms of dsRNA) was mixed with 10 micrograms of protamine(protamine-RNA ratio: 2-1).

Three minutes after adding protamine to the RNA, 1 ml of Ringer lactatewas added and the whole solution transferred to a transparent cuvette.The cuvette was placed in a Malvern Zetasizer and the size of theparticles was measured as well as the Polydispersity. In general, goodpolydispersity (i.e. homogeneity of the particles) is bellow 0.1. Thesoftware also gives a “pass” or “fail” quality factor which relates tothe quality of the measurement. A “fail” criteria means that theparticle size distribution is heterogenous or that there are aggregatesfor example. Each experiment was repeated several times and the medianvalues are reported in the below table. When some conditions ledalternatively to a “pass” or “fail” quality factor, “Pass/Fail” isgiven.

Median Size

Water 25 mM NaCl 75 mM NaCl 125 mM NaCl P-R18 334.3 281.3 554.7 1112.7P-HMW 342.3 318 413 597 P-LMW 14147.5 2530.1 1298.7 4407 P-R18-HMW 328.8343 403.3 370 P-R18-LMW 305.8 307.3 669.9 13908.3

Median Polydispersity

Water 25 mM NaCl 75 mM NaCl 125 mM NaCl P-R18 0.06 0.06 0.18 0.26 P-HMW0.75 0.77 1 1 P-LMW 1 1 0.76 1 P-R18-HMW 0.15 0.18 0.4 1 P-R18-LMW 0.050.09 0.45 1

Quality Factor

Water 25 mM NaCl 75 mM NaCl 125 mM NaCl P-R18 Pass Pass Pass Pass P-HMWFail Fail Fail Fail P-LMW Fail Fail Fail Fail P-R18-HMW Pass PassPass/Fail Pass/Fail P-R18-LMW Pass Pass Pass Fail

As found earlier, when ssRNA (RNA18) and protamine are diluted in lowsalt solutions (less than 125 mM NaCl), homogenous nanoparticles areobtained. Homogeneity (polydispersity) is best when the saltconcentration in the diluting solution is 25 mM NaCl or bellow. It isdemonstrated here that dsRNA (both HMW and LMW) when mixed withprotamine fails to give particles of good quality even when reagents arediluted with low salt solutions (high polydispersity and “fail” qualityfactor). However, unexpectedly and surprisingly, when dsRNA is mixedwith ssRNA (RNA18), the addition of protamine allows to generatenanoparticles of good quality in particular when reagents are diluted inpure water or 25 mM NaCl. When mixed with RNA18, LMW dsRNA tends toprovide more homogenous (polydispersity bellow 0.1) particles than HMWdsRNA mixed with RNA18.

RNA and protamine were diluted to 1 mg/ml using pure water. 5 microgramsof RNA (ssRNA:RNA18 or dsRNA or a mixture of 2.5 micrograms of ssRNAwith 2.5 micrograms of dsRNA) were mixed with 10 micrograms ofprotamine. Three minutes later the solutions were centrifuged 3 min at10,000 rpm in a table top centrifuge. The supernatants were then removedcarefully with a yellow tip attached to a micropipette. The particles(pellets) were resuspended in 10 microliters of water containing 0.5%sodium dodecyl sulfate (SDS) and 2 microliters of 10 mg/ml proteinase Kwere added. All mixtures were incubated at 37° C. for 30 minutes so thatprotamine is digested and RNA molecules released free. Then, 3microliters of loading buffer containing 0.5% SDS were added to allsamples. The formulations were loaded on a 2% native agarose gel andmigrated at 2V/cm during one to two hours. The gel on the left of FIG. 1shows an example of the results obtained with HMW dsRNA and the gel onthe right shows an example of the results obtained with LMW dsRNA. “R18”is ssRNA alone, “HMW” is high molecular weight dsRNA, “LMW” is lowmolecular weight dsRNA, “R18+HMW” is a mixture of RNA18 and HMW dsRNA,“R18+LMVV” is a mixture of RNA18 and LMW dsRNA. It is demonstrated thatboth ssRNA and dsRNA alone or mixed together get trapped in theparticles (found in the pellets); see FIG. 1. Thus, when ssRNA and dsRNAare premixed, the addition of protamine allows to generate chimericprotamine/ssRNA/dsRNA particles containing both the dsRNA and the ssRNA.

Example 3 Immunostimulation Capacity of Protamine/ssRNA/dsRNA Particlesas Reflected by Interferon-alpha Production

PBMCs from a healthy human donor were prepared using Ficoll® gradientseparation. They were then washed with PBS and resuspended at 5 millionper ml in RPMI with 10% fetal calf serum plus penicillin andstreptomycin. Two hundred microliters (1 million of cells) were added inwells from a 96 well plate on top of

-   4 micrograms of protamine (“Prot”)-   2 micrograms of ssRNA, oligonucleotide RNA18 (“R18”)-   2 micrograms of High Molecular Weight dsRNA (“HMW”)-   4 micrograms of protamine mixed with 2 micrograms of ssRNA    (“Prot-R18”)-   4 micrograms of protamine mixed with 2 micrograms of High Molecular    Weight dsRNA (“Prot-HMW”)-   4 micrograms of protamine mixed with 1 microgram of ssRNA and 1    microgram of High Molecular Weight dsRNA (“Prot-R18-HMW”)-   2 micrograms of Low Molecular Weight dsRNA (“LMW”)-   4 micrograms of protamine mixed with 2 micrograms of Low Molecular    Weight dsRNA (“Prot-LMW”)-   4 micrograms of protamine mixed with 1 microgram of ssRNA and 1    microgram of Low Molecular Weight dsRNA (“Prot-R18-LMW”)

As negative control, PBMCs were cultured alone.

These preparations were incubated for 18-24 hours at 37° C. with 5% CO2.Then, the supernatants of the cultures were collected. The content ofIFN-alpha in these supernatants was evaluated using 20 microliters ofsupernatants and ELISA kits from Bender. The results are presented inFIG. 2 in pg/ml in the cell culture supernatant. They demonstrate thatchimeric particles containing ssRNA and dsRNA (high or low molecularweight) are immunostimulating. In general, interferon-alpha productionwas superior using stimulation with chimeric particles (ssRNA+dsRNA)than when using particles contained only ssRNA. The addition ofprotamine on dsRNA was not increasing (and rather decreasing the minimalinterferon-alpha induction observed in some experiments by naked dsRNA)its immunostimulating activity as judged by interferon-alpha production.Thus it is surprising that chimeric particles containing both ssRNA anddsRNA are capable to induce high interferon-alpha production.

Example 4 Immunostimulation Capacity of Protamine/ssRNA/dsRNA Particlesas Reflected by Interferon-beta Production

PBMCs from a healthy human were prepared using Ficoll® gradientseparation. They were then washed with PBS and resuspended at 5 millionper ml in RPMI with 10% fetal calf serum plus penicillin andstreptomycin. Two hundred microliters (1 million of cells) were added inwells from a 96 well plate on top of:

-   4 micrograms of protamine (“Prot”)-   2 micrograms of ssRNA, oligonucleotide RNA18 (“R18”)-   2 micrograms of High Molecular Weight dsRNA (“HMW”)-   4 micrograms of protamine mixed with 2 micrograms of ssRNA    (“Prot-R18”)-   4 micrograms of protamine mixed with 2 micrograms of High Molecular    Weight dsRNA (“Prot-HMW”)-   4 micrograms of protamine mixed with 1 microgram of ssRNA and 1    microgram of High Molecular Weight dsRNA (“Prot-R18-HMW”)-   2 micrograms of Low Molecular Weight dsRNA (“LMW”)-   4 micrograms of protamine mixed with 2 micrograms of Low Molecular    Weight dsRNA (“Prot-LMW”)-   4 micrograms of protamine mixed with 1 microgram of ssRNA and 1    microgram of Low Molecular Weight dsRNA (“Prot-R18-LMW”)

As negative control, PBMCs were cultured alone.

These preparations were incubated for 18-24 hours at 37° C. with 5% CO2.Then, the supernatants of the culture were collected. The content ofinterferon-beta in these supernatants was evaluated using 100microliters of supernatants and ELISA kits from eBioscience. The resultsare presented in FIG. 3 in International Units (IU)/ml in the cellculture supernatant. They demonstrate that chimeric particles containingssRNA and dsRNA (high or low molecular weight) are immunostimulating. Ingeneral, interferon-beta production was superior using stimulation withchimeric particles (ssRNA+dsRNA) than when using particles containingonly ssRNA. The addition of protamine on dsRNA was not increasing butrather decreasing its immunostimulating activity as judged byinterferon-beta production. Thus it is surprising that chimericparticles containing both ssRNA and dsRNA are capable to induce highinterferon-beta production.

Example 5 Immunostimulation Capacity of Protamine/ssRNA/dsRNA Particlesas Reflected by Interferon-alpha Production Using DifferentProtamine/Total RNA Ratios

PBMCs from a healthy human were prepared using Ficoll® gradientseparation. They were then washed with PBS and resuspended at 5 millionper ml in RPMI with 10% fetal calf serum plus penicillin andstreptomycin. Two hundred microliters (1 million of cells) were added inwells from a 96 well plate on top of:

-   1 microgram of ssRNA (RNA18) and 1 microgram of High Molecular    Weight dsRNA (“0 for 1”)-   2 micrograms of protamine mixed with 1 microgram of ssRNA (RNA18)    and 1 microgram of High Molecular Weight dsRNA (“1 for 1”)-   4 micrograms of protamine mixed with 1 microgram of ssRNA (RNA18)    and 1 microgram of High Molecular Weight dsRNA (“2 for 1”)-   8 micrograms of protamine mixed with 1 microgram of ssRNA (RNA18)    and 1 microgram of High Molecular Weight dsRNA (“4 for 1”)

These preparations were incubated for 18-24 hours at 37° C. with 5% CO2.Then, the supernatants of the culture were collected. The content ofinterferon-alpha in these supernatants was evaluated using respectively20 microliters of supernatants and the ELISA kit from Bender. Theresults are presented in FIG. 4 in pg/ml in the cell culturesupernatant. They demonstrate that as shown earlier, addition ofprotamine on a mixture of ssRNA and dsRNA allows stimulation of immunecells at protamine-RNA mass ratios from 1-1 up to 4-1.

Example 6 Synergistic Immunostimulating Activity of ssRNA and dsRNA asFar as Interferon-alpha Production by Plasmacytoid DC is ConcernedDepends on Third Cell Type(s)

PBMCs from a healthy human were prepared using Ficoll® gradientseparation. They were then washed with PBS and resuspended at 20 millionper ml in RPMI with 10% fetal calf serum plus penicillin andstreptomycin. Half a ml of cells was incubated with pDC specificmagnetic beads (positive sorting using BDCA-4 beads from Myltenyi) andrun on magnetic column. After washing, the BDCA-4-positive enrichedfraction was recovered. In this cell population, pDCs (BDCA-2 positive)represent more than 50% (in initial whole PBMCs, pDCs represent lessthan 1% of the cells). Recovered cells were diluted in 1.2 ml ofcomplete medium and two hundred microliters of this cell suspension wereadded to the particles formed in the well of a 96 well plate by mixing:

-   -   2 micrograms of ssRNA (RNA18) with 8 micrograms of protamine        (both reagents at 1 mg/ml diluted in pure water). (“Prot-R18”)    -   2 micrograms of dsRNA (high molecular weight poly (I:C)) with 8        micrograms of protamine (both reagents at 1 mg/ml diluted in        pure water). (“Prot-HMW”)    -   1 microgram of ssRNA (RNA18) and 1 microgram of dsRNA        (poly(I:C)) with 8 micrograms of protamine (all reagents at 1        mg/ml diluted in pure water) (“Prot-R18-HMW”)

The negative control is the same pDC-enriched cell population culturedin the presence of 8 micrograms of protamine (“Prot”).

These preparations were incubated for 18-24 hours at 37° C. with 5% CO2.Then, the supernatants of the culture were collected. The content ofIFN-alpha in these supernatants was evaluated using 20 microliters ofsupernatants and ELISA kits from Bender. The results are presented inFIG. 5 in pg/ml in the cell culture supernatant. They demonstrate thatthe high interferon-alpha production in PBMCs induced by chimericparticles containing 1 microgram of ssRNA and 1 microgram of dsRNA isnot obtained in enriched pDCs cell suspension. Protamine-based particlescontaining 2 micrograms of ssRNA are superior in stimulating sorted pDCscompared to chimeric particles containing 1 microgram of ssRNA and 1microgram of dsRNA. Thus, the capacity of the protamine/ssRNA/dsRNAparticles to induce high interferon-alpha production in PBMC dependsprobably on the stimulation by those chimeric particles of other cellsin addition to pDCs. Those cells may produce interferon-alpha or theymay produce mediators that further activate pDCs, enhancing theirinterferon-alpha production.

Example 7 Immunostimulating Capacity of Protamine/ssRNA/dsRNA ParticlesVersus Protamine/ssRNA Mixed with Protamine/dsRNA as Reflected byInterferon-alpha Production

PBMCs from a healthy human donor were prepared using Ficoll® gradientseparation. They were then washed with PBS and resuspended at 5 millionper ml in RPMI with 10% fetal calf serum plus penicillin andstreptomycin. Two hundred microliters (1 million of cells) were added inwells from a 96 well plate on top of

-   2 or 4 micrograms of protamine (“2micProt” and “4micProt”,    respectively)-   2 micrograms of ssRNA, oligonucleotide RNA18 (“2micR18”)-   2 micrograms of Low Molecular Weight dsRNA (“2micLMW”)-   1 microgram of ssRNA, oligonucleotide RNA18 mixed with 1 microgram    of Low Molecular Weight dsRNA (“1micR18+1micLMW”)-   4 micrograms of protamine mixed with 2 micrograms of ssRNA    (“4micProt+2micR18”)-   4 micrograms of protamine mixed with 2 micrograms of Low Molecular    Weight dsRNA (“4micProt+2micLMW”)-   4 micrograms of protamine mixed with 1 microgram of R18 combined    with 1 microgram of Low Molecular Weight dsRNA    (“4micProt+1micR18+1micLMVV”)

A mixture of two independent formulations(“2micProt+1micR18+2micProt+1micLMVV”): the first one containing 2microliters of protamine mixed with 1 microgram of single stranded RNAand the second one containing 2 microliters of protamine mixed with 1microgram of dsRNA.

As negative control, PBMCs were cultured alone.

These preparations were incubated for 18 hours at 37° C. with 5% CO2.Then, the supernatants of the cultures were collected. The content ofIFN-alpha in these supernatants was evaluated using 20 microliters ofsupernatants and ELISA kits from Bender. The results are presented inFIG. 6 in pg/ml in the cell culture supernatant. They demonstrate thatchimeric particles containing both ssRNA and dsRNA are moreimmunostimulating than a mixture of two formulations containingprotamine+ssRNA and protamine+dsRNA. Thus chimeric protamine-basedparticles containing both ssRNA and dsRNA are capable to induce higherinterferon-alpha production than a combination of independentprotamine-ssRNA and protamine-dsRNA formulations.

The foregoing description and examples have been set forth merely toillustrate the invention and are not intended to be limiting. Sincemodifications of the disclosed embodiments incorporating the spirit andsubstance of the invention may occur to persons skilled in the art, theinvention should be construed broadly to include all variations fallingwithin the scope of the appended claims and equivalents thereof.Furthermore, the teachings and disclosures of ail references citedherein are expressly incorporated in their entireties by reference.

The invention claimed is:
 1. A method for stimulating the immune systemof a subject comprising administering to the subject a pharmaceuticalcomposition comprising a chimeric particle and optionally one or morepharmaceutically acceptable carriers, diluents and/or excipients, thechimeric particle comprising single stranded RNA (ssRNA) and doublestranded RNA (dsRNA), wherein the RNA in the particle is associated withat least one cationic agent, wherein the at least one cationic agentcomprises protamine, wherein the dsRNA comprises two RNA moleculescapable to fully or partially hybridize together, and wherein thechimeric particle is formed by the addition of the at least one cationicagent to premixed ssRNA and dsRNA.
 2. The method of claim 1, wherein thechimeric particle has a diameter in the range of from about 50 nm toabout 1000 nm.
 3. The method of claim 1, wherein the protamine forms acomplex with and/or encloses said RNA.
 4. The method of claim 1, whereinthe at least one cationic agent consists of protamine.
 5. The method ofclaim 1, wherein the protamine: RNA (ssRNA+dsRNA) weight ratio is from16:1 to 1:2, preferably from 4:1 to 1:2.
 6. The method of claim 1,wherein the ssRNA contains at least one U nucleotide and/or at least oneG nucleotide.
 7. The method of claim 1, wherein the ssRNA is anoligonucleotide of from 6 to 100 nucleotides or an mRNA of from 50 to10,000 nucleotides.
 8. The method of claim 1, wherein the ssRNA is anoligonucleotide having the sequence according to SEQ ID NO:
 1. 9. Themethod of claim 1, wherein the strands of the dsRNA are in average from6 to 8000 nucleotides in length.
 10. The method of claim 1, wherein thedsRNA is polyinosinic-polycytidylic acid (poly(I:C)).
 11. The method ofclaim 1, wherein the pharmaceutical composition is co-administered withat least one antigen.